CC BY
4УДК. 582.232:635.64:546.17
Sergiu N. Dobrojan 1, Galina N. Dobrojan 2
1,2 Scientific Research Laboratory, Algology Vasile Salaru", Moldova State University,
(https://usm.md/)
Corresponding author: Dobrojan Sergiu Nicolaevich, sergiudobrojan84@yahoo.com
THE PROPERTY OF CYANOBACTERIA TO QUANTITATIVELY REGULATE THE NITROGEN CONTENT OF WATER AND SOIL
Abstract.The article presents the results of experiments conducted with nitrogen-fixing cyanobacteria Nostoc gelatinosum Schousboe ex Bornet & Flahault, Nostoc flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kützing ex Bornet & Flahault in laboratory conditions (in the aquatic nutrient medium nitrogen-free and in soil), as well as in greenhouses and open ground, aimed at the analysis of nitrogen changes that occur when introducing their biomass. The research results showed that nitrogen-fixing cyanobacteria have the property of "quantitative autoregulation" of nitrogen content in soil and water and contribute to maintaining its ecological balance.
Keywords: cyanobacteria, nitrogen, self-regulation, soil and water
Сергей Николаевич Доброжан1, Галина Николаевна Доброжан2
1,2Научно-исследовательская лаборатория „Альгология Василе Салару", Молдавский
государственный университет, (https://usm.md/)
*Автор, ответственный за переписку: Доброжан Серджиу Николаевич,
sergiudobrojan84@yahoo.com
СВОЙСТВО ЦИАНОБАКТЕРИЙ КОЛИЧЕСТВЕННО РЕГУЛИРОВАТЬ СОДЕРЖАНИЕ АЗОТА В ВОДЕ И ПОЧВЕ
Аннотация. В статье представлены результаты экспериментов, проведенных с азотфиксирующими цианобактериями Nostoc gelatinosum Schousboe ex Bornet & Flahault, Nostoc flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry и Cylindrospermum majus Kützing ex Bornet & Flahault в лабораторных условиях (на водных питательных среды, без азота и на почвы), а также в теплицах и открытого грунта, направленных на анализ изменений азота, образующихся при введении их биомассы. Результаты исследований показали, что азотфиксирующие цианобактерии обладают свойством «количественной авторегуляции» содержания азота в почве и воде и способствуют поддержанию его экологического баланса.
Ключевые слова: цианобактерии, азот, саморегуляция, почва и вода
Introduction. Cyanobacteria play a key role in the biogenic migration of nitrogen from the Earth's surface, which contributes to the maintenance of life on our planet [1]. Cyanobacteria are cosmopolitan, hardy, with a major ability to reproduce, and some of them have the "phenomenal" biological ability to fix atmospheric nitrogen in both aquatic and terrestrial ecosystems in all regions [4].
Atmospheric nitrogen fixed by cyanobacteria is important for the Earth's ecosystems because due to this process the environment accumulates about four times more nitrogen than the amount received from the atmospher [7]. In the most extreme areas, such as the Arctic region, the nitrogen fixed by cyanobacteria compensates for its lack of soil and at the same time these
8
© Dobrojan S.N., Dobrojan G.N., 2022
organisms play a key role in the development of the primary successions of terrestrial ecosystems [8].
Biological nitrogen fixed (BNF) by cyanobacteria is important for the efficient functioning of aquatic ecosystems. Multiple studies have shown that cyanobacteria fix a much higher amount of atmospheric nitrogen in aquatic than in terrestrial ecosystems, and that this process intensifies when the ratio of N:P in water is lower. Atmospheric nitrogen fixed by cyanobacteria directly limits the productivity, composition, dynamics and diversity of many ecosystems by modifying them [3, 6]. When the minor amount of nitrogen contributes to the reduction of phytoplankton productivity, nitrogen-fixing cyanobacteria activate their functions and make up for the deficit with nitrogen fixed in the atmosphere.
It is now well known that cyanobacteria contribute to the accumulation of nitrogen in terrestrial and aquatic ecosystems, largely due to their ability to BNF. However, it is not clear whether these organisms contribute to maintaining the balance of nitrogen in aquatic and terrestrial ecosystems and whether they can be considered as "self-regulators" of this element. This problem is especially important for the biogeochemical analysis of the Earth's ecosystems. We believe that the lack of information on organisms that have the property of quantitative ecological "self-regulation" of nitrogen balance in aquatic and terrestrial ecosystems is the main reason that led us to conduct this research.
Thus, the purpose of this research is to present and explain the hypothesis regarding the property of nitrogen-fixing cyanobacteria of quantitative "self-regulation" of nitrogen in terrestrial and aquatic medium and maintaining its balance.
Materials and methods. In order to establish the quantitative "self-regulatory" property of nitrogen by nitrogen-fixing cyanobacteria, experiments were performed in laboratory conditions on liquid nutrient medium (nitrogen-free) and in soil, as well as in greenhouse conditions when cultivating crops and in open fields.
Experimented cyanobacteria - the experiments included pure unialgal strains of the following species of cyanobacteria: Nostoc gelatinosum Schousboe ex Bornet & Flahault, Nostoc flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kützing ex Bornet & Flahault. The cyanobacterial strains were isolated from the soils of the Republic of Moldova and are in the collection of the Scientific Research Laboratory "Algology Vasile Salaru" of the Moldova State University.
Conditions for conducting experiments - the experiments started in the field were carried out in the central region of the Republic of Moldova, during the years 2014-2020, in the experimental greenhouses for growing tomatoes and cucumbers (equipped with irrigation, adjustable temperature and humidity) and in the open field, without irrigation, when cultivating sunflower.
Cyanobacteria cultivation - to obtain the biomass applied in experiments, the cyanobacteria were cultured, according to the periodic method, on the Drew nutrient medium, preventively sterilized (by exposure to ultraviolet lamp). In laboratory experiments to study the dynamics of nitrogen in water, was used the nitrogen-free culture medium Drew whose composition is described in the literature [2]. The cyanobacteria were inoculated at a dose of 0,4 g/l, being cultured according to the periodic method.
Quantitative determination of nitrogen - experiments performed on liquid nutrients were determined by the forms of nitrogen NH4+, NO3-, NO2- and N of algal biomass, using classical spectrophotometric methods. The determination of total N in the soil was performed by spectrophotometric method using Nessler reagent [2, 4, 9].
Nitrogen from the nutrient medium was determined by the sum N-NH4++N-NO3-+ N-NO2-. Total nitrogen was calculated by summing the N-total biomass of algae (mgN * algal biomass g) + N-NH4++N-NO3-+N-NO2-(from the nutrient medium). The nitrogen eliminated in nutritive medium was calculated using the formula N.el (%) = (Ntm * 100) / (Nt-N0). Where: Ntm - total nitrogen in nutritive medium, Nt - total nitrogen (algal cells + Ntm); No-total nitrogen from inoculum cells.
Results and discussion
Numerous scientific studies conducted so far, which have focused on the analysis of the dynamics of nitrogen modification in the cultivation of nitrogen-fixing cyanobacteria, have shown their indisputable ability to fix and accumulate nitrogen in nitrogen-free nutrients, but also in their use in quality of biofertilizer for the cultivation of crop plants. However, in our view, they have not shown that nitrogen-fixing cyanobacteria have the property of quantitative "self-regulation" of nitrogen in both aquatic and terrestrial medium. The results of the many experiments presented in this paper demonstrate this finding. Thus, to demonstrate the role of nitrogen-fixing cyanobacteria in the quantitative "self-regulation" of nitrogen in water is presented the results obtained from the periodical cultivation of pure strains of cyanobacteria on the Drew nutrient medium (which is free
30 20 10
initial
3-day 6-day 9-day 12-day
initial
3-day 6-day 9-day 12-day
• N. gelatinosum
■ Cyl. majus
N. flagelliforme
N. gelatinosum
■ Cyl. majus
N. flagelliforme
4
0
0
of N and very poor microelements including Mo), and in order to establish this specific legitimacy for the soil, experiments were carried out in laboratory and field conditions by administering the biomass of nitrogen-fixing cyanobacteria on the soil without planting the plants and on the soils involved in agriculture in the cultivation of the crop plants. As it is known, cyanobacteria fix atmospheric nitrogen in water in the form of NH4+ ions after which it is transformed into NO3-. Thus, we analyzed the dynamics of changes in these ions in the cultivation of cyanobacteria on a nutrient medium over a period of 12 days, which ensures the exponential phase of biomass growth.
A B
Fig. 1. Dynamics of modification NH4+ ion (A) and NO3- ion (B) when cultivating nitrogen-fixing cyanobacteria Nostoc gelatinosum Schousboe ex Bornet & Flahault, N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kützing ex Bornet & Flahault, mg/l
As can be seen from the results shown in fig. 1, NH4+ ions tend to increase major on the 3rd day (between 2,34-2,78 mg/l) followed by a slight decrease on the 6th day, after which their concentration increases to on the 9th day (reaching 3,3-3,6 mg/l), and on the 12th day there is a pronounced decrease. Respectively, when analyzing this indicator we can conclude that the experienced cyanobacteria have the property of fixing atmospheric nitrogen in the form of ammonium ions, within the limits of the algal population and their consumption, if there is a surplus in the nutritive medium. This indicates that these organisms have the capacity to regulate the nitrogen in the aqueous medium and to keep it within the necessary limits. Nitrate ions are increasing until the 9th day in all the experimented variants, and on the 12th day their quantity decreases, so it was manifested in ammonium ions. Thus, we can conclude that on the 12th day the amount of NO3- and NH4+ was much higher than the need of the algal population and was activated the mechanism of quantitative "self-regulation" of nitrogen in water. We would like to mention that during the experiments in the nutritive medium no nitrite ions were identified.
120 100 80 60 40 20 0
» N. gelatinosum »Cyl. majus N. flagelliforme
25 20 15 10 5 0
initial 3-day
^^"N. gelatinosum ■
6-day 9-day 12-day
™Cyl. majus N. flagelliforme
A B
Fig. 2. The amount of atmospheric nitrogen fixed (A, mg/l) and that eliminated in the nutritive medium (B,%) when cultivating nitrogen-fixing cyanobacteria Nostoc gelatinosum Schousboe ex Bornet & Flahault, N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kützing ex Bornet & Flahault
The analysis of the changes of the atmospheric nitrogen fixed and eliminated in the nutritive medium of cultivation of the researched cyanobacteria clearly shows the tendency of fixation and consumption of nitrogen. This property is common to all researched cyanobacteria, but the period of its manifestation differs depending on the species. Thus, for example, the population of the species Cylindrospermum majus Kützing ex Bornet & Flahault it fixes atmospheric nitrogen continuously until the 6th day, after which it consumes the fixed nitrogen, and then on the 12th day the process of BNF is initiated again. In populations of Nostoc gelatinosum Schousboe ex Bornet & Flahault and N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry BNF is produced until the 3rd day, after which the fixed nitrogen is consumed on the 6th day, on the 9th day the process is started again, and on the 12th day attests to nitrogen consumption. The same oscillations, characterized by the elimination of atmospheric nitrogen and its consumption, are attested in the analysis of the process of elimination of fixed atmospheric nitrogen (fig. 2).
initial
3-day
6-day
9-day
12-day
I N. gelatinosum Cyl. majus I N. flagelliforme
Fig 3. Biomass of the cyanobacteria population Nostoc gelatinosum Schousboe ex Bornet & Flahault, N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kützing ex Bornet & Flahault, g/l
Biomass of cyanobacterial populations Nostoc gelatinosum Schousboe ex Bornet & Flahault, N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kützing ex Bornet & Flahault is linearly increasing from inoculation to the 12th day of the experiment, but the amount of atmospheric nitrogen fixed and eliminated in the nutritive medium, as I mentioned, varied from one period to another. This indicates that even if the population of the cyanobacteria studied is increasing, the fixed atmospheric nitrogen does not exceed the limits of its need (being consumed in case of surplus and fixed in case of lack). The fact that BNF in cyanobacteria is not influenced by the amount of biomass was also established in the research conducted by Grimm N.B. and Petrone K.C., who concluded that the consumption of Anabaena
5
4
3
2
1
0
cyanobacterial biomass by phytophagous fish in the lotic ecosystem does not influence the BNF's [5]. Thus, we can conclude that cyanobacterial populations Nostoc gelatinosum Schousboe ex Bornet & Flahault, N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kützing ex Bornet & Flahault have the property of quantitatively regulating nitrogen in the nutritive medium and respectively maintaining its balance. This process is subject to a specific physiological control that allows nitrogen to be maintained within the allowed natural limits.
In order to determine whether this legality is also observed in the case of soil, several experiments were performed (in laboratory conditions, greenhouse and open field) where the biomass of cyanobacteria was administered.
0,8 0,6 0,4 0,2 0
Initial
15-day
30-day
■ N. gelatinosum
■ Cyl. majus
45-day
N. flagelliforme
A - 20%
0,5
Initial 15-day 30-day 45-day
"N. gelatinosum Cyl. majus N. flagelliforme
B-40%
0,5
0,5
initial 15-day 30-day 45-day
"N. gelatinosum ^^^^»Cyl. majus ^^^^»N. flagelliforme
initial 15-day 30-day 45-day
"N. gelatinosum ^^^^»Cyl. majus ^^^^»N. flagelliforme
C -60% D-80%
Fig. 4. Dynamics of the modification of total nitrogen in the soil with varying humidity at the administration of cyanobacteria (soil moisture A-20%; B-40%; C-60%; D-80%), %
Laboratory experiments with cyanobacterial biomass administration Nostoc gelatinosum Schousboe ex Bornet & Flahault, N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kützing ex Bornet & Flahault, the results of which are shown in fig. 4, denotes that cyanobacteria fix atmospheric nitrogen up to a certain limit which is dependent on the humidity conditions after which its concentration is reduced, this process is dynamic. The results of these experiments allow us to see the observance of the same legitimacy of "self-regulation" of nitrogen content in the soil by the researched cyanobacteria.
0,8 0,6 0,4 0,2 0
initial 15 - day
9 N. gelatinosum Cyl. majus
A
30- day 45-day ^N. flagelliforme
2 1,5 1 0,5 0
1 day 15 day 30 day 45 day 60 day 75 day
• N. flagelliforme
■ N. gelatinosum
B
Cyl. Licheniforme
1
0
1
1
0
0
Fig. 5. Changes in total soil nitrogen when administering cyanobacterial biomass Nostoc gelatinosum Schousboe ex Bornet & Flahault, N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kutzing ex Bornet & Flahault in the cultivation of tomatoes (A) and cucumbers (B) in greenhouse conditions, %
When administering cyanobacterial biomass Nostoc gelatinosum Schousboe ex Bornet & Flahault, N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kutzing ex Bornet & Flahault the same legitimacy was observed when growing tomatoes and cucumbers in greenhouse conditions. The amount of atmospheric nitrogen accumulated in the soil differs depending on the species administered, the plants grown and the period of monitoring, but it is certain that they fix the nitrogen up to a certain amount (which varies depending on several factors) after which, if this amount it is sufficient for the ecosystem, then its consumption mechanism is triggered, and if it is deficient, is fixed nitrogen from the atmosphere. In the variants with the administration of cyanobacteria to the cultivation of tomatoes, they contributed to the accumulation of nitrogen in the soil until the 30th day of the experiment, although as major consumers of nitrogen were the tomato seedlings, after which the nitrogen decreased to the 45th. day. In the experiment with the administration of biomass in the cultivation of cucumbers, the highest amount of nitrogen in the soil was attested on the 15th day (in the variants with administration of cyanobacteria of the genus Nostoc), and in the variant with the administration of biomass of the species Cylindrospermum majus the largest quantities were attested only on the 45th day (fig. 5).
0,6
0,4 0,2 0
initial 15 - day 30- day 45-day
9 N. gelatinosum Ш N. flagelliforme > Cy. Lichaeniformes
Fig. 6.
Changes in
total soil nitrogen when administering cyanobacterial biomass Nostoc gelatinosum Schousboe ex Bornet & Flahault, N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kützing ex Bornet & Flahault when cultivating sunflower, %
In order to verify that the identified legitimacy is specific and in open conditions, the biomass of the cyanobacteria experienced was administered in the cultivation of sunflower in the open field. The results shown in fig. 6 allow us to find that the same legitimacy of quantitative "self-regulation" of nitrogen in the soil achieved by the experienced nitrogen-fixing cyanobacteria and of maintaining the balance of nitrogen in the soil ecosystem.
Conclusions. Based on the results obtained, we can conclude that nitrogen-fixing cyanobacteria Nostoc gelatinosum Schousboe ex Bornet & Flahault, N. flagelliforme Harvey ex Molinari, Calvo-Pérez & Guiry and Cylindrospermum majus Kützing ex Bornet & Flahault possesses the property of quantitative self-regulation of nitrogen in soil and water. Thus, we can establish that nitrogen-fixing cyanobacteria have the function of balancing atmospheric nitrogen in soil and water and probably have a key role in ensuring the edaphic and aquatic climax.
REFERENCES
1. Dobrojan S., Çalaru V., Çalaru V., Melnic V. Dobrojan G. Cultivarea algelor: monografie. Chi§inäu: CEP USM, 2012. 173 p.
2. Grigheli Gh., §alaru V., Jigäu Gh., Stasiev G., Galburä O. Analiza chimicä a calitä^ii apei. Chi§inäu: CEP USM. 2006. 113 p.
3. Howarth R.W., Marino R., Cole JJ. Nitrogen fixation in freshwater, estuarine, and marine ecosystems. 2. Biogeochemical controls // Limonol. Oceanogr. - 1988. N. 33. - P. 688-701.
4. Peter M. Vitousek P.V., Cassman K., Cleveland C., Crews T., Field C. B., Grimm N.B., Howarth R.W., Marina R., Martinelli L., Rastetter E.B., Sprent J.I. Towards an ecological understanding of biological nitrogen fixation // Biogeochemistry. - 2002. N. 57/58. - P. 1-45.
5. Sandu M., Lozan R., Tan^a A., Ropot V. Metode de instruire privind controlul calitatii apei. Chi§inau „Ericon SRL", 2010. 173 p.
6. Smith V.H., Bennett S.J. Nitrogen: Phosphorus supply ration and phytoplankton community structure in lakes // Arch. Hydrobiol. N. 146. - P. 37-53.
7. Solheim B., Zielke Matthias, Bjerke J. W., Rozema J. Effects of enhanced UV-B radiation on nitrogen fixation in arctic ecosystems // Plant Ecology. - 2006. N. 182. - P. 109 -118.
8. Zielke M., Ekker A.S., Olsen R.A., Spjelkavik S. and Solheim B. The influence of abiotic factors on biological nitrogen fixation in different types of vegetation in the High Arctic, Svalbard // Arct. Antarct. Alp. Res. - 2002. Vol. 34. - P. 293 - 299.
9. Воскресенская О.Л., Алябышева Е.А., Половникова М.Г. Большой практикум по биоэкологии. Ч. 1: учеб. Пособие. Йошкар-Ола: Мар. гос. ун-т., 2006. 107 c.
Information about the authors
S.N. Dobrojan— PhD in biology, assoc. prof.
G.N. Dobrojan — master of ecology
Информация об авторах
С.Н. Доброжан — кандидат биологических наук, доц. профессор
Г.Н. Доброжан — магистр экологии
